Composite materials comprising amyloid fibrils and nanoparticulate nutritional minerals

ABSTRACT

The present invention relates to the use of amyloid fibrils for providing nutritional minerals to the human body, particularly as a component in a food product, a dietary supplement or in a pharmaceutical product. The invention further provides for new compositions comprising composite materials of amyloid fibrils and nutritional minerals. The invention further relates to the use of such composite materials in the treatment of diseases and disorders and to the fortification of food and dietary supplements with nutritional minerals.

This application is a national phase of International Application No.PCT/EP2018/056023 filed Mar. 12, 2018 and published in the Englishlanguage, which claims priority to European Application No. 17160624.7filed Mar. 13, 2017, both of which are incorporated herein by reference.

The present invention relates to the use of amyloid fibrils forproviding nutritional minerals to the human body, particularly as acomponent in a food product, a dietary supplement or in a pharmaceuticalproduct. The invention further provides for new compositions comprisingcomposite materials of amyloid fibrils and nutritional minerals. Theinvention further relates to the use of such composite materials in thetreatment of diseases and disorders and to the fortification of food anddietary supplements with nutritional minerals.

Composite materials of Fe3O4 and beta-lactoglobulin amyloid fibrils(fibBLG) are a known; their synthesis and magnetic behaviour isdescribed in Bolisetty et al (ACS Nano, 2013, 7, 6146). Also, compositematerials of Gold and fibBLG are a known; their synthesis and shapedarticles comprising them are described in Li et al (WO2014/124546).These documents are silent about any use in the context of oraladministration or feeding to humans.

It is well known that a balanced diet is essential in maintaining goodhealth. Hence, the nutritional value of foods is an important aspectthat should be considered especially with respect to metal intake. Foodfortification is a well-established strategy to address this issue.Particularly iron as a nutritional metal is a challenge for foodfortification. Hurrell et al (Heal. Promot. 2002, 1, 806) identify thechallenges of iron fortification, namely (1) finding an iron compoundthat is adequately absorbed but causes no sensory changes to the foodvehicle; and (2) overcoming the inhibitory effect on iron absorption ofdietary components. Further, Huber et al (Small, 2005, 1, 482) discussesapplications of iron nanoparticles, particularly pointing out thedifficulties in handling, due to its extreme reactivity. Typically, ironcompounds used for oral supplementation are limited by low absorption ofthe iron (resulting in poor efficacy) and/or show gastric irritation(resulting in poor compliance). Also, Grage et al (WO2013/010966)discuss that fortification of food with micronutrients such as iron isnot a straightforward procedure. They propose encapsulate embedded in apolysaccaharide phase, said encapsulate comprising gelled proteinsaggregates of 1-5000 micron and containing a micronutritient. Althoughsuitable, such encapsulates are difficult to manufacture and handle.Further, they are limited in the choice of micronutrient and in theirapplications.

Thus, it is an object of the present invention to mitigate at least someof these drawbacks of the state of the art. In particular, it is an aimof the present invention to provide for a versatile platform technologyin food fortification.

These objectives are achieved by the use of amyloid fibrils as definedin claim 1, composite materials as defined in claim 2 and compositionsas defined in claim 10. Further aspects of the invention are disclosedin the specification and independent claims, preferred embodiments aredisclosed in the specification and the dependent claims.

The present invention will be described in more detail below, referringto the first, the second and the third aspect of the invention. Thefirst aspect is directed to new uses of amyloid fibrils and compositematerials comprising amyloid fibrils including their manufacturing. Thesecond aspect is directed to composite materials comprising amyloidfibrils and nutritional minerals in food products, dietary supplementsand pharmaceutical compositions. The third aspect is directed topharmaceutical uses of such composite materials.

Unless otherwise stated, the following definitions shall apply in thisspecification:

It is understood that the various embodiments, preferences and ranges asprovided/disclosed in this specification may be combined at will.Further, depending of the specific embodiment, selected definitions,embodiments or ranges may not apply.

The terms “a,” “an,” “the” and similar terms used in the context of thepresent invention (especially in the context of the claims) are to beconstrued to cover both the singular and plural unless otherwiseindicated herein or clearly contradicted by the context. The terms“including”, “containing” and “comprising” are used herein in theiropen, non-limiting sense.

As used herein, the term “Food Fortification” refers to the practice ofdeliberately increasing the content of an essential micronutrient,particularly nutritional minerals in a food product irrespective ofwhether these micronutrients were originally in the food beforeprocessing or not. By doing so, the nutritional quality of the food isimproved and a health benefit is typically provided for.

As used herein the term “minerals” is used in the context of nutrition,i.e. “minerals essential for mammals, particularly humans”. In thecontext of nutrition, a mineral is a compound comprising one or morechemical elements required as an essential nutrient by the organism toperform functions necessary for life. The term minerals shall include“major minerals” and “trace minerals”, the latter also referred to as“trace elements”. The term major minerals particularly includescompounds comprising the chemical elements calcium, phosphorus,potassium, sodium, and magnesium. The term trace minerals particularlyincludes compounds comprising the chemical elements iron, cobalt,copper, zinc, manganese, molybdenum, iodine, and selenium. In thecontext of this invention “major minerals” and “trace minerals” are alsocollectively termed “nutritional minerals”.

The present invention will be better understood by reference to thefigures.

FIG. 1 shows TEM images of the materials described herein.

a. Amyloid fibrils were produced by heating the 2 wt % of purifiedbeta-lactoglobulin (BLG) protein monomer (pH 2) at 90° C. for 5 h.(scale bar is 100 nm)

b. Iron nanoparticles were synthesized onto amyloid fibrils by in situchemical reduction of FeCl₃.6H₂O to obtain iron-BLG fibrils (i.e thecomposite materials comprising amyloid fibrils and nanoparticulatemineral compounds located on the surface of said amyloid fibrils). 0.45wt % of amyloid fibrils was mixed with 0.015M FeCl₃.6H₂O salt solutions.Fe III ions binding to amyloid fibrils were chemically reduced by NaBH₄.(scale bar is 100 nm)

c. In-vitro acidic/enzymatic digestion of iron-BLG fibrils. Bydecreasing the pH value to 1.2 at 37° C. for 20 min, the iron particlesare readily dissolved and only fibrils are detected (top image, scalebar is 200 nm). Fibrils are digested by pepsin at 37° C. for 1 hourresulting in iron nanoparticles aggregation. (bottom image, scale bar is200 nm).

FIG. 2 shows in vivo study results and sensory performance.

a. Relative bioavailability (RBV %) with confidence intervals (CI) forpowder (left) and liquid compounds (right) against FeSO₄ (100%).Light-grey: Fe-Nano, Dark-Grey: iron-BLG fibrils. No statisticaldifference in RBV was detected between compounds or when compared toFeSO₄ in both powder and liquid form (p>0.05).

b. Color change of iron-containing compounds in powder form in selectedfood matrices at 2.5 mg iron/100 g food matrix. y-axis: Absolute colorchange, ΔE*_(ab)±sd, of 2 replicates is given at 120 min against thenon-fortified matrix. x-axis from left to right: chocolate milk,raspberry yoghurt milk, banana milk, cereal based infant formula,fruit-based infant food.

Black: FeSO₄; light-grey: Fe-Nano, dark-grey: iron-BLG fibrils. Thedetection limit is indicated; iron-BLG fibrils outperform Fe-Nano andFeSO₄.

c. Turbidity of iron-containing compounds in liquid form, compared infish source at 25 mg iron/100 ml fish sauce. Δ Turbidity is relative toFormazin standard solution (FNU) and against the non-fortified fishsauce. Black: FeSO₄; white: Fe pyrophosphate, dark-grey: iron-BLGfibrils. Iron nano compounds could not be tested for turbidity as theyrapidly sediment in liquids.

In more general terms, in a first aspect, the invention provides for theuse of amyloid fibrils for providing nutritional minerals to the humanbody.

Advantageously, these amyloid fibrils are present in the form of acomposite material, said composite material comprising (a) amyloidfibrils and (b) one or more nanoparticulate minerals located on thesurface of said amyloid fibrils. Advantageously, these amyloid fibrilsare a component in a food product, a dietary supplement or in apharmaceutical product.

This aspect of the invention shall be explained in further detail below:

Amyloid Fibrils (a):

The term “amyloid fibrils” is generally known in the field to describe aspecific type of protein aggregates and particularly describes fibrilsmade by proteins or peptides prevalently found in beta-sheet secondarystructure. Accordingly, the term amyloid fibrils excludes nativeproteins. Amyloid fibrils made of pathological proteins are known fortheir association with neurodegenerative disorders, see e.g. Viles et al(Coordination chemistry review, 256, 2012, 2271) and Ghani et al (Int JPept Res Ther 23, 2017. 393).

It was surprisingly found that amyloid fibrils are efficient carriersfor nutritional minerals, particularly for iron fortification and/orzinc fortification. Particularly suitable are amyloid fibrils fromβ-lactoglobulin, an inexpensive milk protein with natural reducingeffects, which proves to act as an anti-oxidizing nanocarrier andcolloidal stabilizer for nanparticulate minerals, such asnanoparticulate iron compounds.

In case of iron, the resulting composite material forms a stableprotein-iron colloidal dispersion undergoing rapid dissolution andrelease of iron ions during acidic and enzymatic in-vitro digestion.Importantly, this composite shows high in-vivo iron bioavailability,equivalent to ferrous sulfate in hemoglobin repletion and stable isotopestudies in rats, but with reduced organoleptic changes in foods, asfurther detailed in the examples provided below. Feeding the rats withthese composite materials did not result in abnormal iron accumulationin any organs, or changes in whole blood glutathione concentrations,inferring their primary safety. Therefore, these nanoparticulateiron-amyloid fibril-composites emerge as highly effective deliverysystems for iron in both solid and liquid matrices.

Advantageously, the amyloid fibrils have high aspect ratio, preferablywith ≤10 nm in diameter and ≥1 μm in length.

Advantageously, the amyloid fibrils have a highly charged surface. Theterm highly charged surfaces is generally known in the field andparticularly describes surfaces showing electrophoretic mobility of theorder 2 μm·cm/V·s at pH 4. Accordingly, amyloid fibrils having anelectrophoretic mobility of the order 1-4 μm·cm/V·s at pH 4 arepreferred.

Advantageously, the amyloid fibrils are obtained from globular proteins,advantageously food grade globular proteins. Globular proteins beingpreferably selected from the group consisting of beta-lactoglobulin(BLG), whey, lysozyme, Bovine serum albumin, soy proteins, ovalbumin,particularly preferably beta-lactoglobulin.

Nanoparticulate Minerals (b):

This term defines both, particle size and chemical composition of theparticles. As the term “nano” implies, particles of 5-100 nm size (asdetermined by microscopy) are particularly useful in the context of thisinvention. The particle size may vary, depending on the mineral. As anexemplary embodiment, for iron, a preferred range of the particles is5-20 nm. As a further exemplary embodiment, for zinc or calcium, apreferred range of the particles is 20-100 nm. Suitable particle sizesmay be determined by the skilled person in routine experiments.

Broadly speaking, any known mineral may be used; preferred arenutritional minerals as defined above. As used herein, the term mineralsshall particularly include compounds selected from the group consistingof salts, oxides and hydroxides.

Nutritional Minerals:

The term is known in the field and discussed above. Accordingly, theterm includes the five major minerals in the human body (calcium,phosphorus, potassium, sodium, and magnesium containing minerals) andthe trace minerals (iron, copper, zinc, manganese, molybdenum, iodine,and selenium containing minerals), particularly Fe, Ca, Mg, and Zncontaining minerals; and very particularly Fe containing minerals.

Nanoparticulate Iron Minerals:

Iron being required e.g. for haemoglobin and for the treatment ofvarious diseases as outlined herein. The term includes oxides,hydroxides and salts of iron. The term includes stoichiometric andnon-stoichiometric compounds. Iron (II) being preferred. Iron (II)compounds relates to chemical entities with iron in the oxidation state+2. As it is known, iron is absorbed in the human intestine only as Fe+2, and is therefore more bioavailable in oxidation states +2. Inaccordance with this invention iron (II) compounds contain at least 50mol-% iron in oxidation state +2, preferably at least 66 mol-%,particularly preferably at least 75 mol % (each in respect to the totalamount of iron). Typically, iron(II) compounds are present as a salt,oxide or hydroxide and combinations thereof. Consequently, the followingexemplary compounds are included: Fe (II) oxides, Fe (II) hydroxides,Fe(II) salts. These nanoparticulate iron (II) compounds may comprise ametallic Fe core, thereby forming nanoparticles of the core-shell type.

Nanoparticulate Calcium Minerals:

Calcium is essential to humans as an important constituent of bones andteeth, and of nerve function. Calcium containing nanoparticles can beattached on amyloid fibrils (particularly BLG fibrils) as well, forminga stable dispersion and potentially deliver calcium in a morebioavailable way with organic compound. As exemplary compounds Calciumcarbonate calcium phosphate, calcium pyrophosphate, calcium citrate, arementioned, particularly suitable is Calcium carbonate.

Nanoparticulate Magnesium Minerals:

Magnesium is essential for humans as it is required for more than 300biochemical reactions in the body that maintain normal nerve, immunesystem, heart, bone and muscle function. Magnesium containingnanoparticles can be generated in situ along the amyloid fibrils(particularly BLG fibrils) forming stable dispersions, allowfortification in liquid and potentially increase the bioavailability. Asexemplary compounds, magnesium oxide and magnesium sulfate arementioned.

Nanoparticulate Zinc Minerals:

Zinc deficiency is a global health problem that causes growthretardation and poor immune function. Zinc containing nanoparticles canbe nucleated in situ on the surface of amyloid fibrils (particularly BLGfibrils). The resulting composite forms stable dispersions, potentiallyincreases the bioavailability. Further, it provides for an antibacterialeffect to thereby extend the fortified foods' shelf life. As exemplarycompounds, zinc oxide and zinc sulfate are mentioned.

Composite Material:

According to the invention, constituents (a) and (b) are in intimatecontact. The individual constituents remain separate and distinct withinthe finished structure, the nanoparticulate mineral (b) being present onthe surface of the amyloid fibrils (a). Without being bound to theory,it is believed that this is ensured by the manufacturing process andinvolves adsorption of the nanoparticles (b) on the fibrils (a) due todifferent surface charge. The composite material may be described aseither of amyloid fibrils, worm-like or spherical nanoclusters (a), ineach case being decorated with nanoparticulate material (b). Thematerial exhibits properties of both, amyloid fibrils andnanoparticulate mineral, and is therefore also termed hybrid material.FIG. 1 shows the structure of the composite material. As can be seen,nanoparticles (b) are predominantly located on the surface of theamyloid fibrils (a), such as at least 90% of the nanoparticles,preferably at least 95% of the nanoparticles are located on the surface(in respect to the total amount of nanoparticles present in thecomposite). Particularly preferably, all nanoparticles (b) are presenton the surface of the amyloid fibrils (a).

It was surprisingly found that constituents (a) and (b) of the compositematerial synergistically interact when administered. The ratio of bothconstituents may vary over a broad range, depending inter alia on thespecific materials and the intended use. Particularly good results areobtained, in case the ratio (a)/(b) is in the range of 20/1 to 1/1(w/w), such as 5/1.

The composite material may be present in various forms. In oneembodiment, the composite material is present as a dry material,preferably-freeze dried material. In one alternative embodiment, thecomposite material is present as a gel, preferably as an aqueous gelcomprising NaCl as an additional component. In one alternativeembodiment, the composite material is present as an aqueous solution,preferably having a pH of 2.7-4 or having a pH of 6-10.

A particularly suitable composite material is obtained when combiningfibBLG (component a) and nanoparticulate iron minerals (compound b). XPSanalysis of this composite material revealed a complex structure ofcomponent (b), showing the presence of Fe(II) oxide and Fe(III)oxi-hydroxide formed around the iron cores, with the residual Fe(II)-Cl₂and Fe(III)-Cl₃ on the nanoparticles surface. As shown in theexperiments provided below, these composite materials show the samebioavailablity as FeSO₄ but with improved sensory performance; furtherthey are colloidally more stable than standard forms of nanosized iron.Without being bound to theory, it is believed that BLG fibrils stabilizeiron in oxidation state (II), which is the more bioavailable oxidationstate. Further, it is believed that the BLG fibrils prevent iron fromcolloidal aggregation. Still further, it is believed that BLG fibrilsalso protect iron against dietary inhibitors, such as phytate,polyphenols and calcium. These beneficial effects make compositematerials comprising (a) BLG fibrils and (b) nano-particulate ironminerals located on the surface of said amyloid fibrils a particularlypromising carrier and delivery system for nanosized iron, thereforeuseful for iron fortification of compositions as described below.

Compositions:

As outlined above, the amyloid fibrils, or composite materialsrespectively, are not directly administered/supplied to a human being.Rather, they may be a component of a wide variety of compositions,including food products, dietary supplements and pharmaceuticalcompositions. Accordingly, the invention also provides for the use ofamyloid fibrils/composite materials as described herein in themanufacturing of a food product, dietary supplement and pharmaceuticalcomposition

Manufacturing:

The composite materials described herein are simple in manufacturing,using starting materials readily available. The ratio of component (a)and (b) can be altered by varying the initial weight fractions of thetwo starting materials. Depending on the nature of compound (b), variousapproaches for manufacturing the composite material are available.Accordingly, the manufacturing is known per se but not yet applied tothe specific starting materials required in the context of the presentinvention. The invention thus provides for a method for manufacturing acomposite material as described herein,

The manufacturing may take place at room temperature, or at slightlyelevated temperatures.

Typically, an aqueous suspension of amyloid fibrils is provided first.The synthesis of amyloid fibrils is a known technology. Suitable is inparticular protein hydrolysis followed by β-sheets driven fibrillation,as described e.g. in Jung et al. (Biomacromolecules. 2008, 9,2477-2486). The self-assembly process is facile and controllable.Typical process parameters include incubating protein solution (e.g. 2wt. % β-lactoglobulin) for a prolonged period of time (e.g. 5 h) underacidic conditions (e.g. pH˜2), low ionic strength (e.g. I≤20 mM), hightemperature (e.g. T˜90° C.). Suitable proteins are food-grade proteins,which are structural stable, wide accessible and inexpensive. Suchproteins allow preparation of amyloid fibrils, such as β-lactoglobulin.Suitable proteins may be selected from the group consisting ofβ-lactoglobulin, lysozyme, ovalbumin, and serum albumins.

For certain applications the obtained composite material may be directlyused. However, the obtained composite material is typically filteredthrough a support material for further use, particularly formanufacturing a composition as defined above.

In one embodiment, the manufacturing of composite material as describedherein comprises the steps of (i) preparation of amyloid fibrils in anaqueous medium, preferably by self-assembly at low pH and low ionicstrength; and (ii) combining these amyloid fibrils with a solutioncomprising metal ions as defined above followed by a reducing agent(preferably NaBH4) to thereby obtain the composite material; and (iii)optionally further treatment. This manufacturing involves the in-situsynthesis of nanoparticulate minerals in step (ii) by providing asuitable solution comprising the metal ions (cations) and optionally theanions required to obtain component (b).

In one alternative embodiment the manufacturing of composite material asdescribed herein comprises the steps of (i) preparation of amyloidfibrils in an aqueous medium, preferably by self-assembly at low pH andlow ionic strength; and (ii) combining these amyloid fibrils with asuspension of pre-fabricated nanoparticulate minerals to thereby obtainthe composite material; and (iii) optionally further treatment. Thismanufacturing involves the ex-situ synthesis of nanoparticulate mineralsand is described e.g. in Bolisetty et al (cited above).

Use:

As outlined above, the composite materials are useful as outlinedherein, particularly as component/additive to food products, to dietarysupplements and to pharmaceuticals.

Accordingly, the invention also provides for the use of amyloid fibrilsas described herein for food fortification and for use of compositematerials as described herein for food fortification.

Accordingly, the invention also provides for a method for providingminerals, particularly nutritional minerals, to the human body, saidmethod comprising the step of administering/feeding amyloid fibrils orcomposite materials (both as described herein) as a component in acomposition selected from the group consisting of food products, dietarysupplements and pharmaceutical products.

In a second aspect, the invention relates to compositions comprising thecomposite materials described herein, said compositions being selectedfrom the group consisting of food products, dietary supplements andpharmaceutical compositions.

This aspect of the invention shall be explained in further detail below.

Food Products:

The term food product is known in the field and describes material,usually of plant or animal origin, that contains essential nutrients(such as carbohydrates, fats, proteins, vitamins, or minerals) and isingested and assimilated by an organism to produce energy, allow forgrowth, and maintain life. Accordingly the term includes drinks andsnack products.

Drinks particularly include flavoured milk products and yoghurtproducts. Snack products particularly include cereal based products andfruit based products. Liquid sauce formulations particularly includefish sauce and soy sauce.

Powder formulations particularly include powdered soup, powderedvegetable sauces, powdered fruit juice and milk powders.

The amount of composite material (b) (as described herein, aspect of theinvention) may vary over a broad range and typically amounts to 1-10 mgnutritional mineral per 100 g food product, preferably 2-3 mgnutritional mineral per 100 g food product.

Dietary Supplements:

The term dietary supplement is known in the field and describes aproduct taken orally that contains one or more ingredients (such asvitamins, minerals, trace elements, fatty acids, amino acids) that areintended to supplement one's diet and are not considered food.Accordingly the term includes tablet formulations, effervescent tabletformulations, powder formulations, as well as gel and liquidformulations. Nutritional doses for the minerals discussed above areknown; a typical nutritional dose of iron is 5-30 mg/day; a typicalnutrition dose for zinc is 5-30 mg/day. The skilled person is in aposition to prepare dietary supplements meeting with these criteria.Accordingly, the amount of composite material (b) (as described herein,1^(st) aspect of the invention) may vary over a broad range andtypically amounts to 1-10 mg nutritional mineral per 100 g dietarysupplement, preferably 2-6 mg nutritional mineral per 100 g dietarysupplement.

Pharmaceutical Compositions:

The term pharmaceutical composition in known in the field and describesany composition suitable for the treatment, prevention or delay ofprogression of a disease in a subject in need thereof. The termparticularly includes formulations adapted for oral administration, e.g.in liquid or solid dosage form. Suitable liquid dosage forms includeoral solutions/suspensions, such as syrup. Suitable solid dosage formsinclude tablets, such as coated or un-coated tablets.

Pharmaceutical doses for the minerals discussed above are known; atypical nutritional dose of iron is 30-300 mg/day; a typical nutritiondose for zinc is 30-300 mg/day. The skilled person is in a position toprepare pharmaceutical compositions meeting with these criteria. Inpharmaceutical compositions, the metal of composite material (b) may bethe only component of the pharmaceutical composition acting as an activeingredient. Alternatively, the pharmaceutical compositions may comprisetwo or more different metals as defined above, or other activeingredients.

In a third aspect, the invention relates to pharmaceutical applicationsof compositions described herein. Suitable dosage regimes and modes ofadministration may be determined by a person skilled in the art.

This aspect of the invention shall be explained in further detail below.

In one embodiment, the invention provides for a composition comprising acomposite material as defined herein (1^(st) aspect of the invention)for use as a pharmaceutical. Suitable pharmaceutical compositions aredescribed herein (2^(nd) aspect of the invention). The use as apharmaceutical includes the therapy, prevention and delay of progressionof a diseases or disorder in a subject in need thereof.

In one embodiment, the invention provides for the use of a compositematerial as defined herein (1^(st) aspect of the invention) for use inthe manufacture of a medicament for the treatment of a diseaseassociated with iron deficiency and/or zinc deficiency.

In one embodiment, the invention provides for a composition comprising acomposite material as defined herein (1^(st) aspect of the invention)for use in the treatment of a disease associated with iron deficiency.Accordingly, the invention also provides for a method for the treatment,prevention or delay of progression of a disease associated with irondeficiency in a subject in need thereof, said method comprising the stepof administering an efficient amount of a composite material asdescribed herein (1^(st) aspect of the invention).

In one embodiment, the invention provides for a composition comprising acomposite material as defined herein (1^(st) aspect of the invention)for use in the treatment of a disease associated with zinc deficiency.Accordingly, the invention also provides for a method for the treatment,prevention or delay of progression of a disease associated with zincdeficiency in a subject in need thereof, said method comprising the stepof administering an efficient amount of a composite material asdescribed herein (1^(st) aspect of the invention).

The term disease associated with iron deficiency is known anparticularly relates to iron deficiency anemia (IDA); the term diseaseassociated with zinc deficiency is also known. Iron and zincdeficiencies are two major health problems, affecting a large part ofthe population of all ages worldwide, especially in developingcountries. It has been estimated that about 2.2 billion people sufferfrom iron deficiency and 2.5 billion people suffer from zinc deficiency.

A sustainable and cost-effective strategy to reduce IDA is ironfortification of foods, but the most bioavailable fortificants causeadverse organoleptic changes in foods, as discussed in Hurrell (citedabove). When iron nanoparticles are added to food matrices, theirtendency to oxidize and rapidly aggregate in solution, as discussed inHuber (cited above), severely limits their use in food fortification.

A sustainable and cost-effective strategy to reduce zinc deficiency iszinc fortification of foods, but currently-available zinc compounds havelow bioavailability and often cause sensory changes in foods. Zinccompounds also have a tendency to aggregate in solutions, which limitstheir use in food fortification.

To further illustrate the invention, the following examples areprovided. These examples are provided with no intend to limit the scopeof the invention.

EXAMPLE 1 Fe-fibBLG 1. Synthesis & Characterization

These hybrids were prepared in-situ (FIG. 1 ab) according to Amar-Yuliet al. (Soft Matter 7, 3348-3357 (2011)). Fe (III) ions strongly bindonto premade 2 wt % fibrils at pH 2. Nanosized iron nanoparticles werethus nucleated on the surface of fibrils by adding sodium borohydride.Transmission electron microscopy (TEM) gives insight into the morphologyof these hybrids before and after nanoparticle composition. Compared tothe typical morphology of BLG fibrils (FIG. 1a ), small (5-20 nm indiameter), spherical, nanoparticles were found to decorate the surfaceof the fibrils (FIG. 1b ). For powder form of the material, the liquidsample was freeze dried.

2. Fe-fibBLG—In Vitro Studies

Acidic dissolution and enzymatic hydrolysis on the material wereperformed separately.

For acidic dissolution, HCl was added to obtain pH=1.2 and the mixturewas stirred for 20 min. at RT. It was found that the iron particles areno longer observed but only the fibrils remained after acidicdissolution (FIG. 1c top).

Similarly, enzymatic hydrolysis was performed on the hybrid material.BLG fibrils were hydrolyzed by pepsin at concentration 2 mg/ml withadditional 150 mM NaCl, shaken at 50 rpm at 37° C. into short peptides.The iron nanoparticles agglomerated, together with protein residues,thereby forming large clumps in one hour as shown in FIG. 1c bottom.

This result indicates that enzymatic hydrolysis of fibrils is slowercompared to the fast acidic dissolution of iron nanoparticles. Thisdifference in digestion kinetics allows the delivery of iron ions priorto the digestion of fibrils at low pH conditions in the stomach, henceavoiding iron-particle aggregation, yet allowing the digestion of boththe organic and inorganic phases via a synergistic acidic-enzymaticdigestion.

3. Fe-fibBLG—In Vivo Studies

3.1 Study design: For 24-25 days 73 rats were made iron deficient(depletion). Over 15 days, 60 rats were fed 3 iron sources incorporatedin the pellet diets with 10 or 20 ppm iron (repletion). Over the entirestudy, 13 and 3 rats received iron deficient (3.9 ppm) and sufficient(35 ppm) diets, respectively.

3.2 The relative bioavailability (RBV) to FeSO₄ of inventive compositesFe-FibBLG in solid form was investigated in-vivo using the hemoglobinrepletion bioassay in rats. Fe nanoparticles (Fe-Nano) synthesized withthe same method but without BLG fibrils were used for comparison.Fe-Nano has similar iron composition as Fe-FibBLG. Fe-FibBLG and Fe-Nanoin powder form showed RBVs of 90% and 95%, respectively (FIG. 2a ). Highbioavailability of the compounds in liquid form was confirmed by astable isotope study, where erythrocyte incorporation of stable isotopesafter gavage administration of ⁵⁷Fe-FibBLG (RBV 99%) and ⁵⁸Fe-Nano (RBV96%) was not significantly different from ⁵⁴FeSO₄. However, when addedto liquids (pH 7), Fe-Nano forms a dark yellow turbid solution withflocculated agglomerates that tend to precipitate, whereas Fe-FibBLGforms a stable transparent dispersion, similar to the freshly made oneat pH 3.

The high RBV of Fe-FibBLG combined with excellent colloidal stabilityshow that the inventive composite material is a versatile and easy wayto fortify iron in liquid matrices.

3.3 Sensory performance of the inventive composites in both powder andliquid forms was further analyzed by the color change in selected foodmatrices. FeSO₄ and iron pyrophosphate (FePP) were used as positive andnegative standards, respectively. Selected food matrices were fortifiedwith iron compounds in solid form at concentrations of 2.5 mg iron/100 gand color change was determined by ΔE (FIG. 2b ), results are shown inFIG. 2b . FePP showed the least color change because of its low watersolubility and tendency to precipitate, which indicates poor absorption.FeSO₄, Fe-FibBLG showed significantly less color changes than FeSO₄ inmost of the matrices, and for chocolate milk this change was below thedetection limit of 5 ΔE*_(ab).

3.4 Stability in liquid matrices: The increase in turbidity afterfortifying fish sauce with 25 mg iron/100 mL and a storage time of 1month at ambient conditions was evaluated.

The results show that Fe-FibBLG causes significant lower turbidity thanFeSO₄ (FIG. 2c ). These results taken together demonstrate thatFe-FibBLG is a promising carrier and delivery system for nanosized iron,as bioavailable as FeSO₄ but with improved sensory performance, andcolloidally more stable than standard forms of nanosized iron.

4. Conclusion

The Fe-fibBLG, available in both powder and liquid forms, was shown tohave excellent physical stability, chemical properties and to undergofast acid dissolution and enzymatic digestion, as demonstrated in-vitro.Fe-fibBLG has a bioavailability equivalent to ferrous sulfate in Hbrepletion and stable isotope studies in rats, but with less organolepticchanges in foods and without any tissue accumulation. Additionally, itstotal iron content of 7.0±0.9% in the powder form (corresponding to˜23.9% weight of BLG), is comparable with current available ironfortificants. Its reducing-antioxidant effects, stability in aqueousdispersion, improved sensory performance and high bioavailability,combined with its low cost, demonstrate that nanoparticulate ironcompounds-amyloid fibril composite materials are suitable ironfortificants in both solid and liquid foods, dietary supplements andpharmaceutical compositions.

EXAMPLE 2 Zn-fibBLG 1. Synthesis & Characterization

In analogy to example 1, a solution of 2 wt % BLG fibrils at pH 2 wasmixed with ZnCl₂. Zinc ions that bound to BLG fibrils were thenchemically reduced by NaBH₄ to nucleate the zinc nanoparticles (mostlymetallic zinc core with ZnO shell) in situ on the fibrils, thus obtainedZn-fibBLG. For powder form of the material, the liquid samples werefirst centrifuged through a membrane with a pore size of 10 kDa toremove excess mineral compounds, and then freeze dried.

2. Conclusion

The experiment clearly demonstrates that a wide variety ofnanoparticulate nutritional mineral compounds may be employed accordingto this invention.

EXAMPLE 3 Fe-fibLysozyme 1. Synthesis & Characterization

in analogy to example 1, a solution of 2 wt % Lysozyme fibrils (obtainedfrom purified lysozyme which was extracted from egg white) at pH 2 wasmixed with FeCl₃. Iron ions that bound to BLG fibrils were thenchemically reduced by NaBH₄ to nucleate the oxidic iron nanoparticles insitu on the fibrils, thus obtained Fe-fibLysozyme. For powder form ofthe material, the liquid sample was freeze dried.

2. Conclusion

The experiment clearly demonstrates that a wide variety of amyloidfibrils may be employed according to this invention.

The invention claimed is:
 1. A method of providing bioavailablenutritional minerals to a human subject, said method comprisingadministering to said subject a composite material comprising (a)amyloid fibrils and (b) one or more nanoparticulate nutritional mineralslocated on the surface of said amyloid fibrils.
 2. The method of claim1, wherein said composite material is a component of a food product, ofa dietary supplement or of a pharmaceutical product.
 3. The methodaccording to claim 1, wherein the amyloid fibrils (a) are selected fromfibrils being ≤10 nm in diameter and ≥1 μm in length; and/or haveelectrophoretic mobilities of the order 1-4 μm·cm/V·s at pH 4; and/orare obtained from milk, egg, soy, serum, mushrooms, and/or insects;and/or are selected from food grade products.
 4. The method according toclaim 1, wherein the amyloid fibrils (a) are obtained from globularproteins.
 5. The method according to claim 1, wherein said minerals (b)are selected from salts, oxides and hydroxides from Fe salts, Fe oxides,Fe hydroxides; Ca salts; Mg salts; and/or Zn salts.
 6. The methodaccording to claim 1, wherein said minerals (b) are iron compoundshaving a particle size in the range of 5-20 nm; and/or are zinccompounds having a particle size in the range of 20-100 nm; and/or arecalcium compounds having a particle size in the range of 20-100 nm (eachas determined by microscopy).
 7. The method according to claim 1,wherein a ratio (a)/(b) is in the range of 20/1 to 1/1 (w/w).
 8. Themethod according to claim 1, wherein the composite material is presentin the form of a dry material; of a gel; of an aqueous solution at pH2-4; or of an aqueous solution at pH 6-10.
 9. The method according toclaim 4, wherein the globular proteins are selected from the groupconsisting of whey, beta-lactoglobulin, lysozyme, Bovine serum albumin,soy proteins, ovalbumin.
 10. The method according to claim 5, whereinthe Fe salts, Fe oxides and Fe hydroxides are selected from compoundscomprising at least 50 mol % Fe (II) in respect to the total amount ofiron, as determined by XPS; the Ca salts are selected from Ca phosphatesand Ca carbonate; the Mg salts are selected from Mg oxide and Mgsulfate; and the Zn salts are selected from Zn oxide and Zn sulfate.